This U01 grant proposal will utilize a new paradigm for early assessment of new vaccine antigen combinations. It will utilize several state-of-the-art immunological technologies to determine the impact of co-administering the novel Na-GST-1 and Na-APR-1 (M74) hookworm vaccines on the immune response to each antigen in a Phase 1 clinical trial conducted in healthy adults living in a hookworm-endemic area of Brazil. The aim is to induce the highest specific neutralizing antibody response to each component of a future co-formulated product, preferably with an IgG1 and IgG3 subclass response with induction of antibodies of high affinity and a significant B-cell memory response. If co-administration does not result in increased safety risk and does not negatively impact the humoral immune response to either of the two antigens, work will proceed to develop a bi-component, coformulated vaccine that will be used in future clinical trials in hookworm-endemic sites. In addition, the proposed clinical trial will serve to optimize the component combinations that will go into the bi-component product early in clinical development, especially the addition of the immunostimulant GLA-AF, thus economizing on time, resources, and the number of subjects required in clinical trials. The result will enable earlier decision-making regarding the optimal components of a new vaccine, in turn accelerating the time to licensure and delivery to the populations in need living in hookworm endemic areas. In most cases, immune responses to vaccines are evaluated by standard technologies such as the indirect enzyme-linked immunosorbent assay (ELISA), virus neutralization, or radiometric antibody-dependent cell-mediated cytotoxicity assay. This is despite the fact that over the last decade several major advances in antibody profiling and B cell techniques have been made in related areas such allergy and atopy, autoimmunity, biotherapeutics, and biodefense. These include the ImmunoCAP method to quantify induced antibody levels, Surface Plasmon Resonance to investigate antibody affinity, and ELISPOT to quantify vaccine-induced specific memory B cells. The objective of this U01 proposal is to apply these advances early in the clinical development of a new vaccine for hookworm, one of the most prevalent and important of the Neglected Tropical Diseases, at a point when they can be used to predict the effect of combining antigens into a single vaccine product before a co-formulated vaccine is developed and tested in larger, more costly Phase 2 and 3 trials.